SUMMARY The goal of this project is to manipulate the piRNA genome defense pathway of the mosquito Aedes aegypti to provide an effective and inherited immune response to arboviruses. This is a novel approach for controlling mosquito-transmitted human pathogenic viruses and addresses a gap in our knowledge of whether viral sequences, like transposon sequences, can provide a genomic and inherited basis for the piRNA silencing pathway. This approach would represent an important advance in mosquito control since it would anchor the basis of viral resistance into the piRNA cluster loci in the genome, which would then be passed down to successive generations. It would also present the opportunity to achieve this resistance without necessarily genetically engineering the mosquito. The manipulation of this pathway to provide heritable resistance to these viruses presents a viable path of exploration due to our findings that the piRNA pathway is expressed in the gastric caeca of the late- instar foregut. We postulate the piRNA system provides resistance to DNA viruses that are pathogenic to mosquitoes with natural selection leading to mosquitoes that acquire piRNA-based resistance to them. We propose that this piRNA-based mechanism of resistance can be, in the first instance, genetically modified to confer resistance against RNA viruses. We will undertake experiments that will identify the piRNA biogenesis genes involved in the somatic piRNA pathway both in late larvae and in the adults, focusing on those tissues in each which encounter either mosquito viral pathogens or human viral pathogens. We will insert sequences from the dengue RNA virus into selected piRNA clusters in the Ae. aegypti genome. We will then determine if piRNAs to dengue virus are generated that recognize and inactivate viral RNAs leading to reduced titers of this virus in infected mosquitoes. We will direct the expression of the necessary components of the piRNA pathway required for viral immunity to those adult tissues in which the arboviruses replicate. All of this research will be conducted in larval and adult Ae. aegypti rather than in cell culture, which will be used only for growing virus and for measuring viral titers in infected female adult mosquitoes. The outcomes would be immediately extendable to human pathogenic viruses such as Zika virus, and to other mosquitoes and pest insects in which the piRNA pathway is somatically expressed. It would also introduce the concept of developing nucleic-acid-based chemical triggers that could result in the natural immunity to non- genetically engineered mosquitoes in the field.